The results show a low proportion of the applied biochar-C mineralised over 12 months in a relatively clay- and C-poor Arenosol , followed by a clay- and C-rich Cambisol (4.6%), and clay-, C- and earthworm-rich Ferralsol (7.0%). The biochar-C mean residence time (MRT), estimated by different models, varied between 44−1079 (Arenosol), 18−172 (Cambisol), and 11−29  years, with the shorter MRT estimated by a one-pool exponential and the longer MRT by an infinite-pool power or a two-pool exponential model. The two-pool model was best fitted to biochar-C mineralisation. The biochar-C recovery in the 12−30 cm soil layer varied from between 1.2% (Arenosol), 2.5−2.7% (Cambisol) and 13.8−15.7%  of the applied biochar-C after 8−12 months. There was a further migration of biochar-C below the 50-cm depth in the Arenosol, as the combined biochar-C recovery in the mineralised pool and soil profile (up to 30 or 50 cm) was 82%, in contrast to 101% in the Cambisol and 104% in the Ferralsol after 12 months. These results indicate that the downward migration of biochar-C was greatest in the Arenosol . Cumulative CO2-C emission from native soil-plant sources was lower (p <0.10) in the biochar-amended vs. non-amended Ferralsol. This field-based study shows that the downward migration of biochar-C exceeded its loss via mineralisation in the Arenosol and Ferralsol, but not in the Cambisol. It is thus important to understand biochar-soil interactions to maximise long-term biochar C sequestration potential in planted soil systems.Pyrogenic carbon (PyC) is an important component of the global soil carbon (C) pool, but its fate, persistence, and loss dynamics in contrasting soils and environments under planted field conditions are poorly understood. To fill this knowledge gap, a  Biochar may be a good surrogate for natural PyC as it has similar, or indeed greater, C sequestration characteristics ; see A two-source C isotope mixing model was used to determine the proportion of biochar-C in total soil C  and then (ii) the proportion of added biochar-C mineralised  with the TCE rate. Since there was little or no relation of the total or biochar-C emission rate with temperature  of C emission rates (R) and the change in temperature (ΔT) and/or moisture (ΔM) was examined. A mixed model was fitted to Δlog10R through the origin (zero intercept) comprising linear effects of ΔT and/or ΔM, with exponentially correlated errors relative to the time interval between measurement points.For determining the relationship of total C and biochar-C emission rates with temperature and/or moisture, a log10 transformation was applied to the C emission data. Average soil temperature data over the 24 h period were used in the relationships. First order differences were computed to remove non-stationarity in the measured variables, that is, the relationship between the change in log10  [LSD test at 5% significance unless stated otherwise.A mixed model analysis of soil C content, C/N ratio, δin plot) . Total bvs. control micro-plots during the first 1.5-months  compared to the control micro-plots  in a combined model of temperature and moisture  or lower  increased soil C content in the 0–10 cm depth, i.e. from 0.7% to 1.9% in the Arenosol, 1.7% to 3.2% in the Cambisol, and 6.3% to 8.6% in the Ferralsol  greater in the biochar-amended soils than the corresponding controls at the 0−8 cm and 8−12 cm soil layers. Whereas, no significant difference was found in the subsoil layers  between the biochar-amended soils than the corresponding controls, except in the Ferralsol where biochar micro-plots had higher total soil C in the 12−20 cm layer at 4 and 8 months than the control (The application of biochar (day zero) significantly  decreased with time  in the Arenosol only. When comparing biochar recovery at 0–12 cm depth during 4 to 12 months (relative to day zero), both Arenosol (72.3–82.5%) and Ferralsol (79.8–85.1%) had a significant decrease, but not the Cambisol (91.4–96.4%). The proportion of biochar-C recovered in the deeper soil depths (below 12 cm) varied with soil and time (p < 0.05), i.e. decreased in the Arenosol and Ferralsol but not in the Cambisol  and moisture . These environmental fluctuations could increase physical weathering and chemical or biological degradation of biochar [vs. ~16%) of nonaromatic C and a lower degree of aromatic condensation (0.3 vs. ~0.8) compared to the wood biochar used by Singh et al. [In this field-based study, the observed mineralisation proportions of the applied biochar-C (between 2.0 and 7.0% over 12 months) were higher than those previously reported (i.e. 0.7 and 2.7% over 12 months) in plant free laboratory studies using comparable il types , 22. Theoduction , 56–58.  biochar , 60. In h et al. .The calculated MRT varied, depending on the model type, from 11−44 years , 29−71 years  or 21−1079 years (infinite-pool power model) . The modCompared to the relatively long MRT  estimated by Fang et al. , this fiOur results show little correlation between biochar-C mineralisation and environmental factors, such as temperature and moisture see . MoreoveWithin the one field environment , we observed 57% lower biochar-C mineralisation in the low C and low clay Aerenosol compared to the relatively C- and clay-rich Cambisol. In this case the results suggest that higher native soil C and plant growth and associated labile-C inputs in the Cambisol would outweigh any protective effects on biochar-C mineralisation that may arise from biochar-clay interactions. Furthermore, across the three soils, we observed a positive correlation between biochar-C mineralisation and total soil C content Fig I in , with thThe greatest proportion of biochar-C mineralised in the Ferralsol among the three soils in the present study contrasted with the results of Fang et al. , 64, whovia mineralisation and recovery in the soil profile (up to 50 cm). These results suggest that biochar is likely to have migrated in the Arenosol through consistent vertical movement to below 50 cm [-1) two weeks after biochar incorporation in the Cobbitty site. The lower concentration of clay in the Arenosol would have resulted in lower organo-biochar-mineral complexes [In the field situation, biochar could be prone to losses from the applied layer via lateral and/or verticle movements. In this study, we minimised the opportunity for lateral surface loss of biochar from the repacked soil by enclosing the microplot area with a ~3 cm raised edge. The results show that only ~82% of the applied biochar-C was recovered in the Arenosol, while 101% and 104% were recovered in the Cambisol and Ferralsol, respectively, after accounting for its loss ow 50 cm , 47. Theomplexes  who founvia ligand exchange) with biochar and organic matter compared to other clay minerals such as kaolinite and smectite [vs. 788 mm) in the Ferralsol (Elliott site) vs. Cambisol (Cobbitty site). The greater earthworm abundance in the biochar-amended vs. the non-amended Ferralsol, or in the other two soils (Cambisol and Arenosol), suggests that wood biochar produced by slow pyrolysis may favour earthworm abundance and activity in a soil system where they exist. Indeed, Van Zwieten et al. [vs. control using an earthworm avoidance assay. These earthworms can ingest finer biochar particles and consequently facilitate their physical mixing and redistribution vertically and/or laterally into the soil profile via bioturbation [The full recovery of the applied biochar-C in the Cambisol and Ferralsol to 30 or 50-cm depth ; Fig 3 ismectite , 67. Howsmectite  and highn et al.  showed surbation .The demonstrated downward migration of biochar needs to be considered when aiming to estimate biochar MRT. The vertical distribution of biochar in the soil may influence its persistence since deeper soil layers generally have lower microbial activity and may have higher clay content  relative2 emission. This is particularly possible in studies where (i) biochar-plant root interactions are non-significant, or deemed to have a non-significant impact on the δ13C signature of soil CO2 efflux [13C signatures of plant-derived and soil C sources are similar, e.g. both sources comprise either C4-dominated [3-dominated δ13C signatures (this study). Although the two-source C isotopic model [The use of a two-source isotopic mixing model to distinguish C sources in a biochar-treated soil is challenging at the natural abundance level, particularly where plant contributions could serve as a third C source . Neverth2 efflux ; and/or ominated , 34 or Cic model , 57, 74 p < 0.01) decreased C emissions from the plant-soil sources in the presence of biochar in the Ferralsol suggest the occurrence of negative priming of native soil or plant-derived C by biochar. This could be due to biochar-induced increases in interactions between native organic matter and reactive clay mineral via ligand exchange in the Ferralsol relative to other soils [via cation bridging. Interestingly, the decreased native C emission (by 1.50 t ha-1) from plant-soil sources in the presence of biochar was larger than the amount (1.37 t ha-1) of biochar-C mineralised over 12 months  in the biochar-amended versus the non-amended Ferralsol on two occasions Fig D in , thus beer soils , 64, 77.2 months . These rnditions .13C-labelled biochar (depleted in δ13C relative to soil) in field settings has provided insights into the persistence and fate of biochar-C, or PyC particularly if biochar is considered as a surrogate material, across contrasting soils and environments. Between 2.0 (Arenosol), 4.6 (Cambisol) and 7.0%  of the biochar-C were mineralised in 12 months. The MRT of biochar, based on the short-term study that principally accounts for mineralisation of relatively labile biochar-C components, has been estimated to vary between 20 and >1000 years, depending soil and model type. The MRT of biochar-C was the longest in the Arenosol and shortest in the Ferralsol. The higher native soil C content, earthworm abundance, N fertilisation and/or pasture growth rate may have contributed to greater biochar-C mineralisation in the Ferralsol (relative to the Arenosol or Cambisol). Our results also suggest that the positive priming effect of high SOC content on biochar-C mineralisation could be offset by the biochar-induced stabilisation of SOC in the Ferralsol. Our findings demonstrate that biochar can migrate vertically into the soil profile (≥ 30–50 cm depth over 12 months) in the order of Arenosol (NSW) > Ferralsol (TAS) > Cambisol (NSW). This is possibly due to direct leaching and infiltration of biochar in soluble or particulate forms, which can be significant in high rainfall areas, in a coarse textured soil (e.g. Arenosol), and/or via ingestion and bioturbation by earthworms. Our study suggests that a careful consideration of site specific characteristics, and natural physical (weathering), chemical (oxidation) and biological (bioturbation) processes, is necessary to optimise biochar production process and application strategy to maximise long-term C sequestration within soil systems. These processes may result in considerable loss of biochar-C via enhanced mineralisation or migration to deeper soil layers or waterways, particularly in a sandy soil such as the Arenosol. It may be prudent to produce biochars for soil application that have high physical strength and hence are resistant to possible slaking, oxidation or biological degradation impacts under natural conditions. The knowledge acquired from this research on the mineralisation, persistence and downward migration of biochar in contrasting soils has relevance for C models to assess its C sequestration potential in managed temperate pasture systems with implications for the global C budget.The novel use of S1 Supporting Information(DOCX)Click here for additional data file.


MicroRNAs are small and non-coding RNA molecules with approximately 22 nt in length that cause inhibition of translation or degradation of mRNA. MiR-155 is a kind of molecule with different functions, such as its role in proliferation, apoptosis, inflammation, differentiation, and immunity. One of its best known functions is apoptosis that affects on caspase-3 activity. The main aim of this study was evaluation of miR-155 inhibition effect on cell proliferation and apoptosis induction in Jurkat cells. In this study, Jurkat cells along with MTT assay were used for evaluation of sensitivity to varied concentrations of miR-155 inhibitor . MiR-155 expression level was analyzed using the quantitative real-time polymerase chain reaction (QRT-PCR). Caspase-3 activity was measured by caspase-3 colorimetric activity assay kit. Unpaired t-test was applied for the analysis of MTT and apoptosis results. Probability of 5% was assumed as statistically significant. According to our results, the use of miR-155 inhibitor increased the activity of caspase-3 by 2 fold in 75 nmol concentration. In this research, we found that the proper increase of miR-155 inhibitor concentration can inhibit miR-155 and consequently increase caspase-3 activity and induce apoptosis in the Jurkat cells leading to cell death ultimately. Apoptosis induction by miRNAs activation or inhibition is probably one of the best and low risk ways of cell death induction in malignancies. Due to role of miR-155 in several cancer cells, it may be used as a therapeutic target in future. MicroRNAs are small, single strand and non-coding molecules with conserved sequence containing approximately 22 nt in length, which are encoded in eukaryote cells. They act as post-transcriptional regulators and cause inhibition of translation or degradation of mRNA -5 by binOne of the best known roles of miRNAs in cancer is down-regulation of miR-15a and miR-16-1 in chronic lymphoid leukemia (CLL) . DeregulApoptosis or programmed cell death is a physiological self-destruction process that is controlled by different genes expression. Proliferation and apoptosis are two essential factors in tissue homeostasis and imbalance of them that may give rise to cancer. Some of miRNAs have roles in tumor through apoptotic or anti-apoptotic effect. Among them miR-155 plays a significant role in cell growth and apoptosis -46. ThisCell culture5 cells per well were seeded in 3 ml complete growth medium in a 6-well culture plate.Jurkat cell line was supplied by the National Cell Bank of Iran, Pasteur Institute, Tehran, Iran. These cells were grown in RPMI1640 medium (Gibco.USA) with 10% fetal Bovine serum (FBS) (Gibco.USA), 1X Penicillin streptomycin antibiotic , and 2mM L-glutamine at 37ºC under a humidified atmosphere consisting of 95% air and 5% CO2 for 7 days. In the day of transfection, trypan-blue was used for cells counting and viability determining. Then 2×10Oligonucleotide sequencesFluorescent inhibitors: anti-miR-155; Fluorescent scramble siRNA . QRT-PCR primers: miR-155 forward primer (5'-TTAATGCTAATCGTGATAGG-3'), U6 forward primer (5'-CTCGCTTCGGCAGCACACATATAC-3'), U6 reverse primer (5'-ACGCTTCACGAATTTGCGTGTC-3').MiRCURY LNA™ microRNA inhibitor and scramble transfection5 cells were plated in 2.5ml medium without antibiotic and plus 10% FBS. In one tube, 100 nmol miR-155 inhibitor was diluted in 250µl OPTIMEM . In another tube, 5µl Lipofectamine 2000 was diluted in 250µl OPTIMEM. After 5minutes incubation, two tubes were combined and incubated for 20 minutes. Finally, this compound was added to the cultured plates. Transfection of the scramble was done by a similar manner. In this study scramble was used for the evaluation of transfection efficiency and non-specific effects of oligonucleotides. All tests were done in duplicate.MiRCURY LNA™ microRNA inhibitor was purchased from Exiqon . Lipofectamine 2000  was used for Jurkat cells transfection according to the manufacturer’s manual. Briefly one day before transfection, 2×10RNA extraction5 cells (from 6-well culture plate) by using Biozol reagent in the third and seventh days post transfection. Briefly, 500μl biozol was added to the cells and followed by phenol/cholorophorm precipitation according to the manufacturer’s instructions. The purity examination of the extracted RNA was done spectrophotometrically at 260/280nm and gel electrophoresis.Total RNA was extracted from the control, scramble and inhibitor treated samples containing 2×10For gaining maximum purity, the extracted RNA was treated by DNAse I (fermentas) based on the manufacturer’s guidelines.MiRNA real time PCRIn this study, the third day extracted RNA was used for miRNA assay. The High-Specificity miRNA QPCR core reagent kit provided the reagents for quantitative PCR amplification of the cDNA templates derived from the miRNAs within a total RNA population. Because of their short length, miRNAs are difficult to detect through standard QRT-PCR protocols. As the first step, the miRNA 1st-strand cDNA synthesis kit  was used to elongate the miRNAs in a polyadenylation reaction. Then the polyadenylated RNA was reverse transcribed into QPCR-ready cDNA. Then the target of interest was amplified and detected using the high-specificity miRNA QPCR core reagent kit . The universal reverse primer served as the downstream primer in this QPCR reaction, so the specificity of the QPCR reaction was provided by the miRNA-specific forward primer. Our forward primer for detection of miR-155 was 5'-TTAATGCTAATCGTGATAGGGGT-3'.MTT assayThe day after transfection, the culture medium was replaced with fresh RPMI containing 10% FBS. The culture plates were incubated for the next day at 37˚C. Then, the cells were treated with MTT solution (5 mg/ml) for 4 h. Thereafter, dimethyl sulfoxide (DMSO) was added to each well and the absorption was measured at the wavelength of 540 nm by a spectrophotometer.Apoptosis assay6 cells were suspended in 100µl of chilled 1X cell lyses buffer and incubated on ice for 10 minutes. Then they were centrifuged for 5 minutes at 10000g. Mixtures assay were prepared in a 96-well plate. 20 µl of 5X assay buffer and 10 µl of caspase-3 substrate (Ac-DEVD-pNA) were added to each test sample. The test samples were incubated for 2 hours at 37C. Then absorbance at 405 nm was measured with a microtiter plate reader. Fold increase in caspase-3 activity was calculated by comparing the OD reading from the induced apoptotic test samples with the level of uninduced control.Apoptosis assay was carried out in duplicate experiments using caspase-3 colorimetric activity assay kit that did not have any positive control (Millipore Company). Briefly miR-155 inhibitor transfections were performed as mentioned above. After 72 h of transfection, 0.5×10Statistical analysisUnpaired t-test was performed to investigate the differences in the obtained results of the three groups. Probability of 5% was assumed as statistically significant.Study subgroups, transfection and miRNA assayThe Jurkat cells were divided into 3 groups: control group, scramble group that was transfected by 75nmol of scramble antisense, as well as test group that was transfected by 25nmol, 50nmol, and 75nmol of miR-155 inhibitor. Transfection efficiency was determined using inverted fluorescence microscope. The transfected cells with at least 80% efficiency were selected for further analysis. QRT-PCR analyses revealed that miR-155 expression was not strongly decreased in the cells transfected by different concentrations of miR-155 inhibitor. No significant differences were observed in miR-155 expression between the control and scramble groups.MicroscopyMiR-155 plays a great role in proliferation of Jurkat cells. In this study, miR-155 inhibitor was used for controlling the cell proliferation. We saw a significant decrease in cell count (by trypan blue staining) and significant cell death increase in the miR-155 inhibitor transfected cells in compare to the control and scramble transfected samples after 48 hours of incubation. Reduction of cell count had direct relation with the added amount of inhibitor. Concentrations of the inhibitors were 25, 50 and 75nmol. By increase of the inhibitor concentration, the cell death rate was increased. By the increase of the inhibitor concentration to 75 nmol, the highest amount of cell death was observed .MTT testCytotoxic effects of different concentrations of miR-155 inhibitor and scramble were evaluated 48 hours after transfection by MTT test. Then the optical densities at 540 nm were obtained for all the three groups: 0.568 for the control group, 0.521 for the scramble group, 0.391, 0.303, and 0.218, respectively for the 25nmol, 50nmol and 75nmol concentrations of miR-155 inhibitor. These results demonstrated that optical density and cell viability were similar in the control and scramble groups .In fact, there was no statistical difference between control and scramble group . However, there were significant differences in the optical density and cell viability between miR-155 inhibitor transfected and control or scramble groups. In other words, there were significant statistical differences among control group; 25nmol, 50 nmol and 75nmol of miR-155 inhibitor transfected groups . As shown in At the 75nmol concentration of miR-155 inhibitor, optical density and cell viability were less than half of their value in the control group. These data indicated that higher concentrations of miR-155 inhibitor had higher toxicity effect on the Jurkat cells and could decrease their viability and proliferation.Caspase-3 activityThe result also showed that through miR-155 inhibition, activity of the caspase-3 was increased. In the control group, caspase-3 activity was low (0.048) and in the scramble group it was 0.05. In the 50 and 75nmol concentration of miR-155 inhibitor, caspase-3 activity was 0.766 and 0.873, respectively. In the 75nmol concentration of the miR-155 inhibitor, activity of the caspase-3 was increased by 2 fold comparing to the control group. There was no statistical difference between control and scramble group .But according to these data, there were statistically significant differences in optical density and caspase-3 activity among the control; scramble and 50nmol, 75nmol of miR-155 inhibitor transfected groups [  respectively] .Authors believed that another method such as Annexin V staining or immune blotting for pro- and mature caspase-3 could have been better method for the demonstration of apoptosis.In most cancers, the use of conventional treatments have many side effects and may well fail to improve disease because of drug resistance . TherefoMany of researches confer that miRNAs can affect all characteristics of the malignant cells including self-sufficiency in growth signals, insensitivity to anti-growth signals, evasion from apoptosis, limitless replicative potential, angiogenesis, and invasion and metastasis . The role of different types of miRNAs has been revealed in the regulation of apoptosis. Some of these miRNAs promote apoptosis, whereas others protect against it. Some of the miRNAs act on the survival or death signaling pathways, whereas others affect cell death through mediating apoptotic signals . Among tMiR-155 is a multifunctional molecule that has an important role in different cancers by several mechanisms . This mithyroid carcinoma , colon cAs mentioned, in our study QRT-PCR analyses revealed that miR-155 expression was not strongly decreased in the cells transfected by different concentrations of miR-155 inhibitor. LNA binds and titrates out the pool of miR-155 but does not cause a degradation in the miRNA.We suggest a co-transfection of cells with a luciferase reporter with and without a miR-155 binding site as a better method to determine whether the active pool of miR-155 decrease in future studies.In this study, we suppressed miR-155 activity by miRCURY LNA™ microRNA inhibitor  and observed that caspase-3 activity increased by 2 folds in 75nmol concentration of inhibitor but Ovcharenko and colleagues showed that miR-155 caused suppression of apoptosis in MDA-MB-453 breast cancer and human T cell leukemia Jurkat cells by blocking caspase-3 activity by identification of genes that differentially influence TRAIL-induced apoptosis through siRNA and miRNA library screening.Totally our results were in consistent with the findings of this group (62). Induction of apoptosis by miRNAs can cause a decrease in proliferation of malignant cells, resulting in the prevention of their growth progression. Due to role of miR-155 in several cancer cells, miR-155 inhibitors may be used as a therapeutic tool in near future.Since miR-155 is one of the most widely up regulated miRNA in multiple human tumors and several reports have indicated that miR-155 inhibition can induce apoptosis, use of patients samples instead of cell lines and models can strongly improve implication of this type studies.Further investigation would be necessary for identification of the exact mechanism through which miR-155 inhibitor influence the growth and proliferation of Jurkat tumor cells.


Cytomegalovirus (CMV) is the most common infectious cause of mental disability in newborns in developed countries. There is an urgent need to establish an early detection and high-throughput screening method for CMV infection using portable detection devices.An antibody analysis method is reported for the detection and identification of CMV antibodies in serum using a biosensor based on high spatial resolution imaging ellipsometry (BIE). CMV antigen (CMV-3A) was immobilized on silicon wafers and used to capture CMV antibodies in serum. An antibody against human immunoglobulin G (anti-IgG) was used to confirm the IgG antibody against CMV captured by the CMV-3A.Our results show that this assay is rapid and specific for the identification of IgG antibody against CMV. Further, patient serum was quantitatively assessed using the standard curve method, and the quantitative results were in agreement with the enzyme-linked immunosorbent assay. The CMV antibody detection sensitivity of BIE reached 0.01 IU/mL.This novel biosensor may be a valuable diagnostic tool for analysis of IgG antibody against CMV during CMV infection screening. CMV is the most common infectious cause of mental disability in newborns in developed countries . Detectie.g., high-throughput multiplexed analysis and quantitative, label-free rapid testing. Previous applications of BIE mainly focus on the biomedical fields [The first biosensor based on imaging ellipsometry (BIE) was developed in 1995 ,16. Compl fields ,18, suchl fields , the detl fields  and antil fields . Thus, tl fields , and thel fields . To dateThe purpose of this study was to detect antibodies against CMV-3A in patient serum using a BIE microarray with CMV-3A, as well as to specifically identify captured IgG antibody against CMV. Antibodies against CMV qualitatively were detected using BIE, and then, goat antibody against human IgG (anti-IgG) was added to the area with the captured CMV antibody to confirm IgG antibody against CMV. A standard curve representing different concentration gradients was also established for the quantitative detection of CMV antibodies. As such, the concentration of CMV antibody in serum was quantitatively detected by BIE and then compared using ELISAs.Silicon wafers were purchased from the General Research Institute for Nonferrous Metals (China). N-hy-droxysuccinimide (NHS) and 1-(3-Dimethyla-minopropyl)-3-ethylcarbodii-mide hydrochloride (EDC), Tween-20, bovine serum albumin (BSA), IgG, and Blocking Buffer  were purchased by Sigma. Anti-IgG and a goat antibody against human IgM (anti-IgM) were purchased from Beijing Bo Sheng Bio-technology Co. Ltd. CMV-3A was purchased from GalaxyBio. The CMV-3A was a fusion of three segments of Pp150, Gp52 and Pp65, which have strong antigenicity epitopes. Fusion of the multi-epitope both enhances the sensitivity/specificity and reduces false negatives. Thus, CMV-3A was used as the ligand in the BIE assay. Purified CMV antibody  was purchased from Antibodies-online.com. Patient serum samples were purchased from QiLu Hospital of Shandong University and clinical information is listed in Table A in TORCH ELISA Kits used to analyze serum samples were purchased from MEDSON Inc. The detection process and analysis were executed according to the manufacturer’s instructions. Microplates were coated with native CMV antigens, highly purified by sucrose gradient centrifugation and inactivated. The solid phase was first treated with the diluted sample, and IgG molecules to CMV were then captured, if present, by the antigens. After washing out all of the other sample components, bound anti-CMV IgG molecules were detected by the addition of specific polyclonal anti-H-IgG antibodies labelled with peroxidase (HRP) in the second incubation. The enzyme captured on the solid phase, acting on the substrate/chromogen mixture, generates an optical signal that is proportional to the amount of anti-CMV IgG antibodies present in the sample. A calibration curve, calibrated against the first W.H.O international standard, makes possible a quantitative determination of the IgG antibody in the patient.2HPO4, and 1.8 mM KH2PO4 (pH 7.3) in deionized water. PBST buffer was prepared with 1% Tween-20 in PBS. CMV-3A (0.1 mg/mL) was prepared with PBST. BSA (10 mg/mL) was prepared with PBST. The blocking reagent was a 1:1 (vol:vol) mixture of 10 mg/mL BSA and 10× Blocking Buffer. Purified CMV antibody, IgG, anti-IgG, and anti-IgM were diluted to a concentration of 0.1 mg/mL with PBST. Serum samples were diluted as indicated with PBST for qualitative or quantitative detection.NE solution was prepared with NHS (0.05 M) and EDC (0.2 M) in deionized water (18.3 MΩcm) from a Milli-Q plus system . Phosphate-buffered saline (PBS) was prepared with 140 mM NaCl, 2.7 mM KCl, 10 mM NaThe BIE combines high spatial resolution imaging ellipsometry FigA in  with a m3. The inlet micro-channels were placed into sample plate, and the outlet micro-channels were connected with pumps  offering negative pressure. The simple channel junctions can be used in serial or parallel formats to simultaneously analyze single or multiple samples.The microfluidic system is used for surface patterning and array production, as well as for solution delivery, ligand immobilization and target capture . The mici.e., I = kd, where I is the light intensity and d is the layer thickness [2)≈K×d, where K = 0.12 [Imaging ellipsometry is a display technique for ultrathin film and surface characterization , which ihickness . Under thickness . There iK = 0.12 . Thus, t2 rectangles and rinsed with deionized water. After soaking in piranha solution  for 30 min to increase the number of silanol groups on the wafer surface, and rinsing with deionized water and ethanol, the wafers were soaked in a mixture of 3-aminopropyltriethoxy-silane (APTES) and absolute ethanol  and incubated for 2 h with gentle agitation. The reaction of APTES with the surface silanol groups resulted in covalent immobilization of-O-Si(OH)2-(CH2)3-NH2, forming a layer of densely packed amino groups on the surface. After rinsing three times with absolute ethanol, the wafers were incubated in a saturated solution of succinic anhydride in ethanol for ≥ 3 h with gentle agitation. The CH2CH2COOCO- group of succinic anhydride reacted with the -NH2 of -O-Si(OH)2-(CH2)3-NH2 group immobilized on the surface, generating carboxyl groups (-(CH2)3NH-CO-(CH2)2-COOH). Once prepared, the wafers were stored in ethanol.Silicon wafers were cut into 20×10 mmThe above-modified silicon wafers were used as the substrate. A piece of silicon wafer was placed on the microfluidic mold of the microfluidic system so that surfaces of the wafers were patterned to form small, regular cells in an array format. Then, the carboxyl groups were activated by pumping 10 μL of NE into each cell at a flow rate of 5 μL/min and passed through the surface of the wafer. In the presence of NHS, EDC transfers carboxyl groups to the Sulfo-NHS ester, which reacts with the amino groups of proteins to immobilize the proteins on surface. Next, the ligand was immobilized: CMV-3A as ligand or probe was pumped into each cell (10 μL per cell at 1 μL/min). Third, blocking occurred by pumping the blocking reagent into each cell (50 μL per cell at 1 μL/min) and passing it through each ligand area. Thus, a sensing array surface with CMV-3A was formed to catch CMV antibodies. Fourth, targets were detected. PBST was used as blank control (50 μL per cell at 1 μL/min) and purified CMV antibody as a positive control (50 μL per cell at 1 μL/min) by pumping them into several of the individual cells. Simultaneously, patient serum samples were also pumped into remaining cells (50 μL per cell at 1 μL/min). The CMV antibodies in the serum were captured when they interacted with the CMV-3A on the sensing surface. All cells were rinsed with PBST (20 μL per cell at 20 μL/min) between every two consecutive operation steps. Finally, the microarray wafers were removed from the microfluidic system. After being rinsed with deionized water and dried with nitrogen, the wafers were analyzed using imaging ellipsometry. If the CMV antibodies in the solution or serum interacted with the CMV-3A on the surface and formed a complex, the layer in that area became thicker. The experimental results were recorded as images in grayscale, and binding of CMV antibodies resulted in a significant increase in grayscale value.To detect the specificity of antibodies against CMV, CMV-3A as ligand was immobilized on two columns . PBST buWe further analyzed the types of CMV antibodies captured by the CMV-3A . In the To establish a calibration curve, a serum sample  of BIE and ELISA was calculated with analysis of correlation coefficient in Microsoft Office Excel . The value of the negative control was 125.9 ± 3.18. The mean grayscale value of the negative control minus the mean grayscale value of the blank control was 8.4 (P = 0.07). This indicated that there were specific interactions between the purified CMV antibody and the CMV-3A. Therefore, CMV antibodies in patient samples could be captured by the CMV-3A immobilized on the substrate.Compared to blank controls areas, the purified CMV antibody and patient serum sample detection areas had markedly thicker films, with the average grayscale value displaying significant increases, while negative control areas did not . The meaP = 2.8×10−5), indicating that CMV antibodies were abundant in the serum samples. This was consistent with the ELISA results . The mean value of the anti-IgM minus the mean grayscale value of the blank control was 8.15 (P = 0.29). These data are indicative of the specific interactions between the anti-IgG and the human IgG immobilized on the chip, which could be used as references to determine whether the antibodies in samples are IgG.When purified human IgG was used as the ligand, the average grayscale value of the anti-IgG detection areas significantly increased, while the anti-IgM detection areas did not (left two columns in P = 0.02). However, the value of the anti-IgM was 154.25 ± 0.25, and the difference was not obvious (P = 0.53). This indicated that IgG CMV antibodies were captured by the chip. For sample No. 940 detection, the value of the anti-IgG was 237.7 ± 11, and the increase was 77.3 (P = 0.01). The result also indicated that the content of IgG in samples No. 948 and 940 was different.Compared to the CMV antibody areas, the anti-IgG detection areas displayed significant increases, while the anti-IgM detection areas did not . For exai.e., ~ 6.1% greater than that of the controls (P = 0.03). Repeating these tests > 10 times, our results demonstrated that the sensitivity of the assay reached 0.01 IU/mL or less. Each variation in grayscale value was linked to a corresponding concentration of the CMV antibody over the range of 0.011–2.725 IU/mL, and the calibration curve is shown in Five concentration gradients of a serum sample  , and is followed by IgG production ,31. LevePresently, most methods mention the relative sensitivity (%) of detection for CMV antibodies ,36, but r-value) and P-value were calculated after excluding the number of severe mutations , r-value = 0.93 and P = 0.8 the statistical results showed more agreement between ELISA and BIE.However, there is a substantial discordance between the ELISA and the BIE in some patients . Samples from these patients displayed stronger signal using ELISA than BIE. In ELISAs, anti-CMV IgG molecules are detected by the addition of polyclonal specific anti-H-IgG antibodies labelled with horseradish peroxidase (HRP). In contrast, the BIE biosensor directly identified the variation of the surface anti-CMV IgG concentration after capturing target without a secondary label. Thus, this label-free method may avoid some interference factor. In addition, the grayscale images offered uniform areas, helping to avoid false positive signals. When the correlation coefficient  Imaging principle [Fig B. Microfluidic system. (a) Internal structure; and (b) Laboratory prototype.rinciple ; and (b)(DOCX)Click here for additional data file.S2 FileContains Table A. Clinical information of CMV patients from QiLu Hospital of Shandong University. Table B. Statistical analysis process of comparison between the ELISA and BIE data.(DOCX)Click here for additional data file.


The results of these tests can be compared with experimental data and allow interpretations with regard to biophysical mechanisms. Infectio also facilitates characterizations of the mode of action of therapeutic agents, such as oncolytic viruses or other infectious or cytotoxic agents.Infectio presents a generalized platform to analyze virus infection spread between cells. It allows the simulation of plaque phenotypes from image-based assays. Viral plaques are the result of virus spreading from primary infected cells to neighboring cells. This is a complex process and involves neighborhood effects at cell-cell contact sites or fluid dynamics in the extracellular medium. Infectio differentiates between two major modes of virus transmission between cells, allowing  http://infectio.github.io/index.html.Viruses spread between cells, tissues, and organisms by cell-free and cell-cell mechanisms, depending on the cell type, the nature of the virus, or the phase of the infection cycle. The mode of viral transmission has a large impact on disease development, the outcome of antiviral therapies or the efficacy of gene therapy protocols. The transmission mode of viruses can be addressed in tissue culture systems using live-cell imaging. Yet even in relatively simple cell cultures, the mechanisms of viral transmission are difficult to distinguish. Here we present a cross-platform software framework called “Infectio,” which is capable of simulating transmission phenotypes in tissue culture of virtually any virus. Infectio can estimate interdependent biological parameters, for example for vaccinia virus infection, and differentiate between cell-cell and cell-free virus spreading. Infectio assists in elucidating virus transmission mechanisms, a feature useful for designing strategies of perturbing or enhancing viral transmission. The complexity of the Infectio software is low compared to that of other software commonly used to quantitate features of cell biological images, which yields stable and relatively error-free output from Infectio. The software is open source (GPLv3 license), and operates on the major platforms . The complete source code can be downloaded from IMPORTANCE Infectio presents a generalized platform to analyze virus infection spread between cells. It allows the simulation of plaque phenotypes from image-based assays. Viral plaques are the result of virus spreading from primary infected cells to neighboring cells. This is a complex process and involves neighborhood effects at cell-cell contact sites or fluid dynamics in the extracellular medium. Infectio differentiates between two major modes of virus transmission between cells, allowing in silico testing of hypotheses about spreading mechanisms of any virus which can be grown in cell cultures, based on experimentally measured parameters, such as infection intensity or cell killing. The results of these tests can be compared with experimental data and allow interpretations with regard to biophysical mechanisms. Infectio also facilitates characterizations of the mode of action of therapeutic agents, such as oncolytic viruses or other infectious or cytotoxic agents. Viruses are ubiquitous and infect all forms of life by mechanisms which are often specific for a certain virus type. Therapeutic interventions can target the entire virus replication cycle to either inhibit virus disease or enhance viral efficacy in therapeutic settings, such as oncolytic virotherapy –6. An ar–7–11–Viruses spread between cells by cell-free and cell-cell contact-dependent mechanisms. This involves lytic or nonlytic egress, superinfection repulsion, syncytium formation, or combinations of such mechanisms –19. An e–An example for complex cell-cell transmission phenotypes is vaccinia virus (VACV), an enveloped brick-shaped particle 360 by 270 by 250 nm in size with a large double-stranded DNA of ~200 kbp , 23. It 26–Virus infectivity and transmission are classically measured by plaque assays in cultured cells , 33. HowA generic virus spreading model should be able to simulate plaque formation by a large variety of viruses. It should simulate different modes of transmission, including cell-free and cell-to-cell routes. Our previously published model for cell-free virus diffusion was a hybrid of CA and partial differential equations discretized using the PSE method , 34. To To implement mechanistic parameters in a black-box abstraction model, we built in an option to make the speed of virus cell-to-cell spread dependent on the production of cell-associated virus by an infected cell and on the microinfectivity of the neighboring cells. Such a detailed model can make predictions about a number of VACV mechanisms that are currently discussed  . This inTo test the ability of Infectio to simulate phenotypes of both cell-free and cell-to-cell virus transmission mechanisms, we used published datasets of spatial dynamics of HAdV and VACV , 31. To 10.1128/mSphere.00078-15.3Movie S1Movie S1, MOV file, 10.7 MB.Addition of an advection term to the model yields simulated comet-shaped virus spread patterns for HAdV. Plotted PSE particles with color-coded strength (virus amount) reveal the concentration field behavior at different speeds of advection. Still images are provided in Copyright © 2016 Yakimovich et al.2016Yakimovich et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00078-15.4Movie S2Movie S2, MOV file, 3.2 MB.Simulated HAdV spreading time-lapse with an advection term. The cellular patterns of HAdV-infected cells closely resemble comets observed in microscopy; here, advection speed was 0.5 µm/s. Still images are provided in Copyright © 2016 Yakimovich et al.2016Yakimovich et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To estimate how cell-free VACV contributes to the spread of infection, we have simulated a combination of cell-to-cell and cell-free virus transmission conditions of different VACV strains. The key parameters were either measured, estimated, or inferred from published datasets of WR and IHD-J strains, and graphical outputs were compared with phenotypes from microscopy experiments . We mimiCondition A mimicked the situation in semisolid medium under the assumption that VACV is too large to freely diffuse in the semisolid, and plaque formation occurs exclusively through cell-to-cell spreading , and the proportion of cell-free virus production for VACV WR was 0.1 instead of 0.01. Still images are provided in Copyright © 2016 Yakimovich et al.2016Yakimovich et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Condition C mimicked the situation in semisolid medium where cell-free VACV-IHD-J is subject to diffusion but not advection . We conc10.1128/mSphere.00078-15.8Movie S6Movie S6, MOV file, 4.6 MB.Simulated time-lapse of VACV WR and VACV IHD-J spread with cell-free spread by diffusion. Still images are provided in Copyright © 2016 Yakimovich et al.2016Yakimovich et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vitro and in silico settings and comparing mutants, strains, and species in plaque formation and infection biology will enable novel insights for disease management and the use of viruses in therapies.The large diversity of pathogens and the difficulty of growing certain viruses imply that a crowdsourcing computational effort could improve the models for virus transmission, for example, by using the most accurate experimental data and the most flexible software implementations. To enhance accessibility and utilize expertise from other researchers, we have deposited the Infectio software on the crowdsourcing platform GitHub . GitHub in silico method contains correlates with the cyclomatic complexity, which is a measure of linearly independent paths through the source code for a program  and undergo a time- and PFU-dependent process of cell-free and cell-associated virus production according to the mathematical model with a ratio of cell-free to cell-associated virus  as discussed below see . CA cell10.1128/mSphere.00078-15.1Figure S1 Figure S1, TIF file, 0.9 MB.Fitting of VACV IHD-J extracellular virus production with a sigmoidal growth model. (A) Fitted curve. (B) Fitting residuals. (C) Fitting parameters and estimation of the goodness of fit. Download Copyright © 2016 Yakimovich et al.2016Yakimovich et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00078-15.2Figure S2 Figure S2, PDF file, 6.4 MB.Complexity metrics arrays for Infectio compared to CellProfiler software. (A) An array of complexity metrics, including help metrics, cyclomatic complexity, counts of commentary lines and total lines computed for Infectio v0.1, v0.2, and v0.3. (B) An array of complexity metrics, including help metrics, cyclomatic complexity, commentaries lines count, and total line count, computed for widely used CellProfiler software. Here, all the values of the metrics were applied with min-max normalization of all values for the entire data set in a comparative manner. Download Copyright © 2016 Yakimovich et al.2016Yakimovich et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Analyses of experimentally determined parameters of viral spreading in plaque assay indicated a contribution of cell-free adenovirus to infection , 31. It http://infectio.github.io or in the project help under http://infectio.github.io/help.html.The algorithmic hybrid implementation of the cellular automaton and particle strength exchange (CA-PSE) methods is largely identical to the one published before . Here, wThe proposed method has been implemented as a software framework in the form of a stand-alone package. The main programming language was MATLAB with a well-structured modular design using MATLAB name spaces, which is reflected, for example, in the folder structures of the source code. Following this approach, it is easy to extend the framework by adding, setting files, or enumerating the model parameter space, each of which is different from a default scenario. Introduction of further model parameters or changes in the logics can be made using setting files. Additionally, to ensure user-friendliness of the software, we implemented a MATLAB GUID-based graphical user interface (GUI). Performance-critical parts were implemented as cross-platform C code, which is integrated using the MATLAB MEX technology. Application performance was further enhanced using parallel computing techniques through the shared memory model based on OpenMP 4.0 extensions. To ensure sufficient performance, the recommended hardware requirements include a high-end multicore workstation, or a multipurpose high-performance computer cluster.The Infectio software is designed to take in a number of custom parameters defined by the end-user based on, e.g., experimental measurements as discussed below. An overview of input parameters, flags, and their default values are provided in To measure the probability of infection by cell-free VACV strains WR and IHD-J, we used the time-lapse titration data from a published data set . From thThe speed of cell-to-cell spread was measured from the speed of radial growth (in micrometers per hour), detected by the emergence of the GFP signal around the initially infected cell. Although this measurement is probably less than the speed of virus transition between cells, we took it as an approximation of virus transmission from the originally infected cell to the neighboring cells.Y is the virus amount, t is time after infection, and “Bottom,” “Top,” “EC50,” and “Slope” are the fitting parameters.VACV-WR produces 10 to 100 times less cell-free virus than IHD-J and 100 to 1000 times less cell-free than cell-associated virus , 31. BotWe compared the output of the model with infection spread of different viruses and obtained closely matching results. Next, we estimated the production rate of cell-associated viruses by conjecturing that VACV-IHD-J produces cell-free virus according to the model we fitted see . We alsokB is Boltzmann’s constant, T is temperature in kelvin, η is the dynamic viscosity of the medium, and r is the spherical radius of the diffusing particle.To estimate the diffusion coefficient we used the Einstein-Stokes relation (2/s. Although VACV particles are barrel shaped and not spherical (As previously shown , the Reypherical , we assu


In the absence of transmission sources in combined clinical PET/MR systems, MR images are used for MR-based attenuation correction (MRAC). The main challenge in MR-AC is to separate the bone and air, as neither have a signal in the MR images. In the attenuation maps supplied by the vendor, a single value is assigned to bone using an ultra-short echo time (UTE) MR sequence. The purpose of this study was to develop a new multi-class segmentation-based MR-AC method, employing Continuous-Bone-using-R2* (MRAC_CBuR2*), and evaluate it on a large patient cohort. 53 [18F]-FDG PET/MR brain patients were included in this study. MRAC was based on an aligned CT , standard MRAC_UTE and MRAC_CBuR2*. Our method segments the air, brain, CSF and soft tissue voxels on the UTE images, and uses a mapping of R2* values to HU to measure the density in bone voxels. Aligned anatomical masks are used to improve accuracy in noisy regions. Region-based analysis was performed using ICBM 2009a brain atlas with anatomical labels pre-defined. Using CBuR2*, 82% of the voxels in the brain are within ±5% of PET_CT, compared to 27% when using UTE. Using our method, there are clear improvements over UTE. The average error over the full brain is 0.8% (±1.7%), compared to -7.1% (±2.4%) in UTE. Of note, the maximum error in the cerebellum is -15% and 7% in UTE and CBuR2*, respectively. The proposed method uses the available UTE images to segment tissue classes, and uses the R2* map to measure a continuous bone signal. The improvement over the vendor provided UTE reduces both the global and local error on the reconstructed PET images.


Staphylococcus pseudintermedius (Sp) are pathogens that cause respiratory disease in dogs. Considering bacterial infections following influenza are a leading cause of illness and death, it is of particular meaning to investigate the interaction between these two pathogens. In this study, BALB/c mice were used as a mouse model to assess whether inoculation with CIV H3N2 followed by S. pseudintermedius 72 h later resulted in exacerbation of disease. Disease was characterized by assessment of body weight loss, titration of virus and bacteria, histopathology, and cytokine production.Canine influenza virus (CIV) and S. pseudintermedius elevated the viral RNA load of CIV in tissues. The histological lesions in the brain, spleen and lung were more severe in the CIV/Sp group than in the singly treated groups. Infection with CIV alone, Sp alone or coinfection stimulated a significantly higher release of cytokines, such as interferon-gamma (IFN)-γ, interleukin 6 (IL)-6, tumor necrosis factor (TNF-α) and lymphotactin (Lptn), than was observed in the mock-infected group (PBS). Moreover, the levels of IFN-γ in the spleen and lung were higher in the CIV/Sp group compared with the CIV-only and Sp-only groups.There was a significantly greater decrease in body weight in the co-infected group compared with the CIV-only and SP-only groups. CIV inoculation increased bacterial colonization, whereas secondary infection with Our findings provide the first demonstration that the secondary infection of mice with Sp leads to increased clinical signs and lesions during canine influenza.The online version of this article (doi:10.1186/s12917-016-0708-6) contains supplementary material, which is available to authorized users. Canine infectious respiratory disease (CIRD) is a common, worldwide disease syndrome, and involves a number of viral and bacterial pathogens. Canine influenza virus (CIV) is one of the viral causes of contagious respiratory disease . In JanuBordetella bronchiseptica, Streptococcus equi subspecies zooepidemicus and Mycoplasma [Staphylococcus pseudintermedius (Sp) was the most frequently isolated bacterial pathogen (24/40) from respiratory cases (unpublished data). This bacterium has been previously misidentified as S. intermedius. The name S. pseudintermedius is now given to the canine-specific strain of S. intermedius [S. pseudintermedius is a common commensal of oral and nasal vacities and the skin in healthy dogs, where it can also cause invasive disease [S. pseudintermedius (MRSP) has emerged as an increasingly important cause of opportunistic infections in dogs [Bacterial pathogens associated with CIRD include coplasma . In humacoplasma , 9. A stermedius . S. pseu disease , 12. Sas disease  isolated disease , 15 and  disease . Further in dogs . It may In humans, co-infections of influenza virus and respiratory bacterial pathogens are common, and bacterial secondary infections have played an important role in influenza virus pandemics , 19. InfAlthough CIV infection is typically self-limited with high-morbidity but low mortality, co-infections with respiratory bacterial pathogens may impact the pathogenicity and course of disease. It will be of particular importance considering the role of secondary bacterial infections as a major complication of influenza in pet clinics. To understand the treatment procedures for fighting viral and secondary bacterial infections, it is important to study the mechanisms involved in the interaction between influenza virus and bacterial organisms. In the present study, we investigated the interactions between CIV and Sp and evaluated pathological lesions in mice.5 plaque forming units (PFU)/ml [9 colony forming units (CFU)/ml was made in phosphate-buffered saline (PBS).The viral strain A/Canine/Jiangsu/06/2010 (H3N2) was used in this study. The viral strain was isolated from nasopharyngeal swabs of dogs with severe respiratory syndrome, which were collected from Animal Clinics of Nanjing Agricultural University in Jiangsu Province of China in 2010. The GenBank accession numbers are JN247615 to JN247623. Plaque assays were performed on monolayers of Madin-Darby canine kidney (MDCK) cells in 12-well tissue culture plates to determine the viral titres [(PFU)/ml . The Sp (PFU)/ml  and sequBALB/c mice  were purchased from the Animal Experiment Centre, Yangzhou University, China. All of the mice were housed in individual compartments in stainless-steel wire cages. Before conducting the study, approval for conducting the experiments was obtained from the Animal Ethics Committee of Nanjing Agricultural University.9 CFU/ml, 50 μl/mouse) and 72 h later inoculated with non-infectious MDCK cell culture supernatant (50 μl/mouse); the mice in group 2 (CIV-only) were intranasally inoculated with PBS (50 μl/mouse) and 72 h later inoculated with CIV ; the mice in group 3 (CIV/Sp) were intranasally inoculated with CIV and 72 h later inoculated with Sp; and the mice in group 4 were inoculated with non-infectious MDCK cell culture supernatant and 72 h later inoculated with PBS. The groups were housed in individual isolation rooms.One hundred mice were randomly divided into four groups with 25 mice per group. The mice in group 1 (Sp-only) were intranasally inoculated with Sp.The lung, spleen and brain tissues from mice belonging to the various groups were harvested at 2 days post Sp challenge in the co-infected group and were fixed in 10 % formalin. After fixation, the tissues were embedded in paraffin wax. Tissue sections with a thickness of 4 μm were stained with haematoxylin and eosin (H&E) and assessed for the degree of inflammation and necrosis. The lungs were assigned a grade 0 to 3 based on the histological characteristics as described by Alymova et al. . A scoreSamples of lung, spleen and brain from each mouse were weighed and homogenized individually in PBS to obtain a 10 % weight-to-volume suspension. The number of CFUs of Sp was determined by plating serial 10-fold dilutions of homogenates or blood on LB agar in duplicate. Therefore, the results are reported as CFU/ml for blood and as CFU/g for the lung, spleen and brain.g for 30 min, and the supernatants were collected for viral RNA extraction using a Virus Nucleic Acid Extraction Kit II . Quantitative RT-PCR (RT-qPCR) was performed to measure the viral loads in the above-mentioned organs and blood samples. The RNA from the samples was reverse-transcribed and run in an ABI 7300 Real-time PCR System using a SYBR1 Premix Ex Taq™  kit . The following primers were designed and used on the matrix gene region: 5′-TCTATCGTCCCATCAGGC/GGTCTTGTCTTTAGCCATTC-3′. Simple Vector pMD19-T  containing the target virus sequence was used as a reference standard. Series of eight 10-fold dilutions equivalent to 1×103-1×1010 copies per reaction were prepared to generate calibration curves and were run in parallel with the test samples [The tissues were homogenized in lysates at a ratio of 1:1 (g/ml) and centrifuged at 10,000  samples .The cytokine level was assessed to determine the correlation between severe disease and inflammatory cytokine production in the mice. The spleen and lung from the mice in all of the groups were homogenized in 1 ml of PBS/1 g of tissues. The homogenates were centrifuged, and the supernatants were frozen at −70 °C until testing. ELISAs for the levels of interferon-gamma (IFN-γ), interleukin 6 (IL-6), lymphotactin (Lptn) and tumor necrosis factor (TNF-α) on the lung and spleen from infected mice were performed using ELISA kits  according to the manufacturer’s instructions.P<0.05 were considered statistically significant, and differences with P<0.01 were considered highly significant.The data were collected and analysed using MS Excel 2010 and SPSS Statistics v20.0 software. The weight loss/gain, bacterial load, viral load and cytokine levels were analysed by analysis of variance (ANOVA) followed by Turkey’s multiple comparison test , 30. DifThe mice showed normal features after bacterial inoculation, but after 18 h post-infection (h.p.i), all the infected mice manifested with lethargy, anorexia, huddling and ruffled fur. It is notable that nervous signs appeared in the mice of SP (2/25) and CIV/Sp (4/25) groups at 20 h.p.i. On 2 d.p.i., this phenomenon was more pronounced. Distinct central nervous disturbances, including bending of their head towards one side and walking in circles, were observed in the Sp and CIV/Sp groups. More severe clinical signs were observed in the CIV/Sp group when the mice were lifted by the tail. Such neurological signs lasted until the end of experiment. But the number of affected mice did not increase. Mice with neurological signs lost more body weight compared with the other mice in the same group. In contrast, the mice in the CIV group did not show any neurological signs, but the signs, such as depression, decreased activity, huddling and ruffled fur, were witnessed in all the infected mice from 24 h.p.i., and lasted until the end of experimentP<0.05) between the sham and CIV groups only at 4 and 5 d.p.i. In contrast, Sp-alone-inoculated mice showed signs of more severe illness, and there was a significant difference between the sham and Sp groups (P<0.001) at each time point. Significant differences were also observed between the sham and CIV/Sp groups (P<0.001) at each time point. Mice coinfected with CIV and Sp lost more body weight compared with the mice in both the CIV-only (P<0.001) and Sp-only (P<0.05 or P<0.01) groups at 2, 3, 4 and 5 d.p.i., indicating that mice coinfected with CIV followed by S. pseudintermedius developed more severe disease.All of the infected groups exhibited a lowered body weight compared with the PBS group Fig. . Mice inP>0.05). However, the bacterial loads in the spleen showed significantly higher level in the CIV/Sp group than in the Sp-only group (P<0.01). At 2 d.p.i., the bacterial loads in the blood, brain, spleen and lung had reached a peak level, and showed a significant higher level in the blood, spleen and lung of the coinfection group compared to the SP-lonely group (P<0.05 or P < 0.01). At 3 d.p.i., bacterial load was significantly higher in the spleen of CIV/Sp group than that in the SP-lonely group (P<0.01), and at 5 d.p.i., the bacterial loads in the blood, brain, spleen and lung were decreased to more than 102, 105, 104 and 104 CFU/g (CFU/ml) in both the two groups, respectively.The bacterial loads in the blood, brain, spleen and lungs of mice infected with bacteria alone and with both virus and bacteria were compared Fig. . At eachRT-qPCR was used to measure the viral loads in the above tissues of mice infected with virus alone and with both virus and bacteria Fig. . At all The extent of histological lesions in the brain, spleen and lungs of mice infected with CIV/Sp and with either Sp or CIV alone was compared at 2 d.p.i. All of the tissues tested in the Sp-only, CIV-only and CIV/Sp groups showed significant lesions, whereas the mice in the PBS group showed no lesions in the lungs, brain and spleen Fig. .Fig. 4HiThe brain of the mice in the CIV-only group exhibited the dissolution of nerve fibres. In some areas, a diffused hyperplasia of microglial cells was noticed. In addition, the disintegration of neuronal cells was observed Fig. . The braP<0.01) and the Sp group (P<0.05).The histopathological scores for the H&E sections of the lungs from mice were determined Fig. . The his-γ, TNF-α, IL-6 and Lptn in the spleen and lung of mice at 1, 3 and 5 d.p.i. were measured by ELISA  between the three infection groups and the control group with the exception of the IFN-γ and IL-6 levels in the spleen between the Sp-only and PBS group and the TNF-α levels of the lung between the CIV-only or CIV/Sp group and the PBS group.The levels of IFNP < 0.01 or P < 0.05). At 1 d.p.i., the IL-6 levels of the spleen and lung showed a significant difference between the Sp and PBS groups. At 5 d.p.i., the IL-6 levels of the spleen in the Sp-only group were significantly higher compared with the levels in the CIV-only and CIV/Sp groups (P<0.01 or P<0.05), whereas there was no significant difference in the IL-6 levels in the lung between either the Sp-only or CIV-only group and the CIV/Sp group. In the case of TNF-α and Lptn, at 1 and 5 d.p.i., the highest levels in the spleen were observed in the Sp-only group, whereas the highest levels in the lung were found in the CIV-only group. There was no significant difference between the Sp-only or CIV-only group and the CIV/Sp group.Compared with the CIV-only and Sp-only groups, at 1, 3 and 5 d.p.i., the levels of IFN-γ in the spleen and lung were higher in the CIV/Sp group, and IFN-γ level of the spleen reached the statistical significance at 1 d.p.i. (Several studies have reported that influenza virus A is extremely contagious to humans and domestic animals, including avian, swine, equine, canine and feline species , and cauS. pneumoniae showed greater bacterial counts after 24 and 48 h of infection [S. aureus 3 and 7 d after influenza virus A infection [Our study demonstrated increased bacterial loads in the blood, brain, spleen and lung of the mice in the CIV/Sp group compared with those in the Sp-only group. The finding is in agreement with observations from an earlier study conducted by Lee et al.  in whichnfection . The natnfection , 36. Bacnfection –39. Prevnfection . Howevernfection , 6.S. pneumoniae and reported that the subsequent S. pneumoniae infection enhances viral release from infected cells. Therefore, we speculate that the observed increase in viral loads after bacterial infection may be due to increased virus release via bacterial proteases. Additionally, we cannot rule out the possible mechanism that elevated viral loads in the presence of bacteria are due to a reduction in viral clearance. Further work is necessary to study the precise mechanism involved.The present study showed that the bacterial loads in the blood, brain, spleen and lungs peaked in Sp-only and CIV/sp groups on d.p.i. 2, indicating that the bacteria can enter the bloodstream, are transported to the other tissues and then pass through the blood–brain barrier into the brain, thus causing severe neurological signs. Distinct central nervous disturbances observed at this time, including bending of the head towards one side and walking in circles, support this hypothesis. More severe neurologic signs were observed in the coinfection group. Interestingly, in addition to an increased bacterial burden, we found that the viral loads in the blood, brain, spleen and lung were augmented following bacterial infection. Smith et al.  used a kThe loads of both CIV and Sp in the coinfected mice were greater compared with those of singly infected mice. These results may help explain the increased severity of the lesions observed in the coinfected mice. Additionally, cytokine production after infection likely plays a role in the pathological changes observed during infection. A previous study reported that an excessive production of TNF-α leads to immunopathology . The stuAlthough mice are extensively used for influenza research, and they can be successfully infected with CIV , 6, the Taken together, the findings presented in this study showed that coinfection with CIV and Sp may worsen clinical signs and lesions in mice. This additive effect supports the need for improving the current strategies for the control of influenza and secondary bacterial infection. Because CIV is a relatively recently described canine pathogen, the available clinical data are relatively limited, and further epidemiological studies on the interactions between this virus and secondary bacterial infection are required.Staphylococcus pseudintermedius; TNF-α, Tumor necrosis factor.ANOVA, Analysis of variance; CFU, Colony forming unit; CIV, Canine influenza virus; ELISA, Enzyme Linked Immunosorbent Assay; IFN-γ, Interferon-gamma; IL-6, Interleukin 6; LPTN, Lymphotactin; MDCK, Madin-Darby canine kidney; PBS, phosphate-buffered saline; PCR, Polymerized chain reaction; PFU, Plague forming unit; RNA, Ribonucleic acid; SP,


The ideal intraocular lens in cases of aphakia without capsular support is debated. Choices include anterior chamber lenses, iris- or scleral-sutured lenses, and iris-claw lenses. Our aim was to report our long-term evaluation of the use of retropupillary implantation of the Artisan iris-claw intraocular lens (RPICIOL) in several aphakic conditions without capsular support.A retrospective analysis of consecutive 320 eyes of 320 patients  without capsular support in which we performed RPICIOL implantation in post-traumatic aphakia , post-cataract surgery aphakia , and in cases in which penetrating keratoplasty was associated with vitrectomy . Either anterior or posterior vitrectomy procedures were performed with 20–, 23-, or 25-gauge techniques for different associated anterior or posterior segment indications. We reviewed the refractive outcome, anatomical outcome, long-term stability of the implants, and possible long-term complications.2) at the end of the follow-up period.The mean patient age was 59.7 years  in group 1; 60.1 years  in group 2; and 65.8 years  in group 3. The mean follow-up time was 5.3 years . At the end of the follow-up period, the mean post-operative best-corrected LogMAR visual acuity was 0.6  in group 1; 0.3  in group 2; and 0.6  in group 3. Disenclavation of RPICIOLs occurred in three cases because of slippage of one of the iris-claw haptics and spontaneous complete posterior dislocation occurred in one case. One case presented with retinal detachment, and no cases of uveitis were observed. Eight cases complained of chronic dull pain, and severe iridodonesis was seen in five cases. One case of post-operative macular edema was observed without post-operative increase in the mean intraocular pressure. There was no statistically different change in the endothelial cell density (cells/mmRPICIOL for secondary implantations is a valid alternative strategy to scleral-fixated or angle-supported IOL implantation. The ideal intraocular lens in cases of aphakia without capsular support is debated. Choices include anterior chamber lenses, iris- or scleral-sutured lenses, and iris-claw lenses, with the latter now being used more commonly. The retropupillary approach for iris-claw intraocular lens (RPICIOL) implantation has recently gained popularity. In 1971, Worst first presented the iris-claw lens  at a meeting in Paris  publisheThe aim of this retrospective study was to evaluate the feasibility and safety of retropupillary implantation of the Artisan iris-claw lens in different aphakic situations without adequate capsular support.This was a retrospective study of RPICIOL implantation in 320 eyes of 320 patients  at the Department of Ophthalmology, Hospital S. Maria delle Croci, Ravenna, Italy from January 2002 to December 2009. All patients gave signed informed consent following a discussion of the details of the intervention and the possible risks. The study followed the guidelines of the Declaration of Helsinki and the study protocol was approved by the committee of medical ethics of the Hospital S. Maria delle Croci, Ravenna, Italy.Group 1: Subjects suffered post-traumatic subluxation or total dislocation of the crystalline lens or ruptured cataractous lens without adequate capsular support, with or without severe lacerations of the iris that required reconstruction. Cases with or without traumatic retinal detachment were included. Aphakia in these cases resulted from the severe intraocular trauma and patients required both anterior and posterior segment reconstruction. All RPICIOL implantations were primary procedures during the main reconstructive procedures.Group 2: Subjects suffered post-cataract surgery aphakia that resulted from intra- or post-operative complications, which included dropped fragments or the whole nucleus of the crystalline lens. Cases with subluxation or total dislocation of the IOL that required both anterior and posterior vitrectomy were also included. RPICIOL implantations were primary procedures during intra-operative complications and secondary procedures following post-operative complications. All were one-step procedures.Group 3: Subjects included cases in which keratoplasty was performed in association with vitrectomy. In these cases, we used a Landers temporary keratoprosthesis  during vitrectomy before completion of the keratoplasty procedure.st, 7th, and 30th post-operative days, and then annually in the follow-up period. At post-operative follow-up assessments, we assessed best-corrected visual acuity using LogMAR, IOP, and anterior segment examination with slit lamp. Pre-operatively, endothelial cell density (cells/mm2) using a non-contact specular microscope , was recorded for all patients in group 1 and group 2, and yearly postoperative endothelial cell density (cells/mm2) was assessed for the same group. Endothelial cell density was also assessed in group 3, but with pre-operative eye bank assessment.Were rubeosis iridis and total aniridia. Pre-operatively, we performed the following investigations: best-corrected visual acuity using a LogMAR chart, keratometry, biometry, and anterior segment examination with slit lamp. Post-operatively, we examined patients on the 1The lens used in this study was the Artisan aphakia IOL  which is a polymethyl methacrylate IOL with an 8.5-mm length, 1.04-mm maximum height, and 5.4-mm optical zone width. The optic power was calculated using the SRK/T formula with the aim of achieving emmetropia. The manufacturer’s recommendation for A constant is 115.0 for implantation above the iris but we used an A constant of 116.5 because we implanted the lens retropupillary.All surgeries were performed by two experienced surgeons under either general or peribulbar anesthesia and both surgeons used the same surgical technique. One corneal paracentesis was performed at the side of the surgeon’s non-dominant hand at either the 3 or 9 o’clock position. Acetylcholine 1 % was injected intracamerally through the paracentesis for miosis, followed by injection of a dispersive cohesive viscoelastic material . A 5.4-mm corneal incision was made at 12 o’clock and the iris-claw IOL was then inserted upside down (with its convex surface placed posteriorly), rotated with an Artisan lens forceps to a horizontal position, and centered on the pupil. The optic of the reversed iris-claw IOL was held securely with forceps. Next, the two haptics were gently slid behind the iris and the optic was lifted slightly forward toward the posterior surface of the iris so that the claw configuration of the haptic could be recognized from above on the iris anterior surface. With the other hand, a long micro-spatula was used through a lateral paracentesis at either 3 or 9 o’clock based on the surgeon’s non-dominant hand, to insert iris tissue into the claw. The second haptic was fixated in the same way, using the same spatula. Care was taken to apply gentle pressure over the slotted center of the lens haptic to enclavate a fair amount of iris tissue to avoid ovalisation of the pupil and to decrease the effect of enclavation on the movement of the pupil. We did not perform peripheral iridectomies in any of the cases. At the end of the procedure, we closed the corneal incision with three simple interrupted 10-0 nylon sutures. In cases that were combined with keratoplasty, and after vitrectomy for posterior segment complications using Landers keratoprosthesis, we implanted the lens using an open-sky technique.We used ophthalmic endoscopy using a fused fiber 30 000-pixel 18G probe containing both the light source and the video input  to evaluate the state of the remaining capsule and to confirm removal of the entire remnant of the capsule to avoid post-operative fibrosis that could affect the stability of the RPICIOL. We also used endoscopy to evaluate and confirm enclavation of an acceptable amount of iris tissue between the iris-claw, and the strength of fixation of the RPICIOL to the posterior surface of the iris.We included 320 eyes of 320 patients and all 320 eyes underwent RPICIOL implantation. Group A included 141 eyes, group B included 122 eyes (primary implantations following intra-operative complications in 76 cases and secondary implantations following post-operative complications in 46 cases), and group 3 included 57 eyes. The mean baseline best-corrected LogMAR visual acuity was 1.0  in group 1, 0.7  in group 2 and 0.8  in group 3. The mean patient age was 59.7 years  in group 1, 60.1 years  in group 2, and 65.8 years  in group 3. The mean follow-up time was 5.3 years . The post-operative residual spherical equivalent error was−1.42 diopters (D) ± 1.22 (D) standard deviation (SD) in group 1, −1.5 ± 1.15 (D) in group 2, and −2.4 ± 2.1 (D) in group 3. At the end of the follow-up period, the mean post-operative best-corrected LogMAR visual acuity was 0.6  in group 1, 0.3  in group 2, and 0.6  in group 3. In all cases, the RPICIOLs were in stable position except for three cases that presented with subluxation because of slippage of one of the iris-claw haptics after a mean follow-up of 12 months . RIPICIOLs were refixated in these three cases. Another case presented with complete spontaneous dislocation of the Artisan lens in the vitreous cavity. This case had post-traumatic aphakia, retinal detachment, and an opaque cornea, which were treated by keratoplasty, 23-gauge vitrectomy, and heavy silicone. After 2 months, spontaneous dislocation occurred after removing the silicone. In this case, the Artisan lens explanted and scleral fixation was performed to avoid the stiff, traumatized iris. One case developed retinal detachment after RPICIOL implantation for post-traumatic aphakia. We saw no cases of uveitis after RPICIOL implantation. Eight cases complained of chronic dull eye pain following RPICIOL implantation, which decreased with time. Three cases complained of blurred vision when leaning forward and five cases showed severe iridodonesis. The mean post-operative intraocular pressure in the three groups was 16.4 ± 3.4 mmHg. Three cases in group 1, two cases in group 2, and two cases in group 3 presented with post-operative increased intraocular pressure (24 ± 4.2 mmHg), which was managed medically with topical anti-glaucoma drugs. We had three cases of cystoid macular edema, verified by Stratus optical coherence tomography, which developed 2 months after RPICIOL implantation and vitrectomy for posterior dislocated IOL. Oval pupil was seen in 5 % (16 eyes) of the patients, especially in association with iridoplasty (11 patients). Pigment dispersion was detected in three eyes in group 1. When 20-gauge vitrectomy was a common procedure, we implanted 16 RPICIOLs in 2 years (2002–2003). When we changed to 23-gauge and 25-gauge vitrectomy, we implanted 304 RPICIOLs in 6 years (2004–2009)  was (2227.4 ± 524.4 SD) and the mean post-operative values were (2170.4 ± 431.7 SD). This difference was not statistically significant (P = 0.96).The pre-operative mean and SD of the endothelial cell density (cells/mmSurgical correction of aphakia without capsular support remains a challenge. Each of the available options has its own risks and complications: transscleral fixation of posterior chamber IOLs is an extremely technically demanding procedure with relatively high risk of intra-operative and post-operative complications and requires a large amount of dissection into the conjunctiva and the sclera , 16. AngTwo studies of RPICIOL implantation showed pigment dispersion as a complication, but this was not seen in several additional studies –12, 14. Although some authors consider that the possibility of total luxation of the RPICIOL to be remote, an intra-operative case has been reported , and we Some believe it is preferable to perform an IOL exchange rather than a new fixation of the same IOL, particularly in cases of traumatically dislocated iris-claw IOLs, because of the considerable alterations on the affected haptics of the IOLs, which might not guarantee a reliable re-enclavation. However, we easily managed our three cases of subluxation by refixation, with good results . We expeOvalisation of the pupil was seen in 5 % of patients and especially in patients who underwent iris reconstruction, as previously reported . In our Rijneveld et al.  found irWe saw elevated IOP in seven cases and all were managed medically; our results were consistent with previous reports , 11. We One case of retinal detachment was reported in our series in a post-traumatic aphakia patient who underwent both RPICIOL and posterior vitrectomy. The retinal detachment could have resulted from the initial disease or the second intervention.Eight cases complained of dull aching eye pain, three cases had blurred vision when leaning forward, and five cases showed severe iridodonesis. We believe that these effects resulted from the weight of the lens itself pushing the iris toward the cornea when patients leaned forward. The same mechanism could cause chronic dull pain.When we shifted to 23-gauge and 25-gauge vitrectomy, RPICIOL implantations for managing aphakia without capsular support increased dramatically in our institution. The high feasibility of the technique allowed us to perform new transconjunctival vitrectomy techniques without the need for invasive techniques related to secondary implantation for aphakia without capsular support.The strengths of this study are the long follow-up and the large number of patients. The limitations of this study are the retrospective design and the lack of comparison with anterior implantation of the iris-claw lens.The complications related to RPICIOL implantation were minimal compared with its benefits. Therefore, using retropupillary implantation of the iris-claw lens for secondary implantations is a valid alternative strategy to the classic scleral-fixed or angle-supported IOL implantation.


Objective. The most effective nonpharmacological treatment for insomnia disorder is cognitive behavioural therapy-insomnia (CBT-i). However CBT-i may not suit everyone. Auricular acupuncture (AA) is a complementary treatment. Studies show that it may alleviate insomnia symptoms. The aim of this randomised controlled study was to compare treatment effects of AA with CBT-i and evaluate symptoms of insomnia severity, anxiety, and depression. Method. Fifty-nine participants, mean age 60.5 years (SD 9.4), with insomnia disorder were randomised to group treatment with AA or CBT-i. Self-report questionnaires, the Insomnia Severity Index (ISI), Dysfunctional Beliefs and Attitudes about Sleep scale (DBAS-16), Epworth Sleepiness Scale (ESS), and Hospital Anxiety and Depression scale (HAD), were collected at baseline, after treatment, and at 6-month follow-up. A series of linear mixed models were performed to examine treatment effect over time between and within the groups. Results. Significant between-group improvements were seen in favour of CBT-i in ISI after treatment and at the 6-month follow-up and in DBAS-16 after treatment. Both groups showed significant within-group postintervention improvements in ISI, and these changes were maintained six months later. The CBT-i group also showed a significant reduction in DBAS-16 after treatment and six months later. Conclusions. Compared to CBT-i, AA, as offered in this study, cannot be considered an effective stand-alone treatment for insomnia disorder. The trial is registered with ClinicalTrials.gov NCT01765959. Insomnia disorder is a common and growing problem, which is approximately twice as common for women as for men . Aside fTreatments for insomnia disorder can be pharmacological, nonpharmacological, or a combination of both. Nonbenzodiazepines, also known as z-drugs, are a common pharmacological treatment of insomnia disorder . AlthougIn the present study, the target population was persons who had used z-drugs for more than six months and experienced an unsatisfactory treatment outcome. Insomnia research over the last two decades shows a trend towards increased nonpharmacological interventions, where behavioural therapies are emphasised . The mosAcupuncture is a nonpharmacological treatment method based on complementary medicine, for which interest has increased over the last years. It has, to some extent, been introduced in the ordinary health care system . AcupuncAA has also been used within psychiatric care for substance dependence e.g., , 27), wh, wh27]),Aim. The primary aim of this study was to compare treatment effects of AA with CBT-i and evaluate symptoms of insomnia severity for short- and long-term perspectives. Symptoms of anxiety and depression were also evaluated.This was a prospective randomised controlled study (RCT) evaluating immediate and long-term treatment effects.Inclusion criteria covered men and women (18–75 years) with insomnia disorder, diagnosed according to the DSM-5 , who hadN = 59) who had had insomnia for more than six months and despite pharmacological treatment had residual insomnia symptoms and wanted to discontinue their medication. They were included between January 2013 and October 2014. The participants' mean age was 60.5 (SD 9.4). Twenty-six participants were retired, twenty-nine were working, and one was studying. Three participants were on long-term sick leave and/or had disability pension and one participant was unemployed.The final sample comprised 50 women and 9 men . The subjects from the outpatient sleep clinic were recruited by the senior author (AM). All were informed that participation was voluntary and that they could withdraw from the study at any time without any negative consequences. Inclusion was made at a clinical visit with AM, an experienced medical sleep specialist, who assessed all subjects before inclusion. At this point the consent form was signed after which the participants were included in the study. After inclusion the participants were randomised to group treatment with AA or CBT-i  were used. Before the needles were inserted the acupuncturists' hands and the participants' outer ears were disinfected with disinfectant solution.The CBT-i group received manual-based group treatment , focusedISI is a seven-item questionnaire assessing the severity of insomnia as regards sleep onset and maintenance of sleep, satisfaction with current sleep patterns, interference with daily functioning, impairment due to sleep problems, and level of distress caused by insomnia disorder. The rating scale is 0–4 for each item with a total score range from 0 to 28. A higher score indicates higher severity of insomnia symptoms . A changPrevious studies confirm an association between the level of insomnia severity and dysfunctional beliefs about sleeplessness . DBAS-16In order to measure daytime sleepiness ESS was used. Eight questions, graded from 0 to 3, describe everyday situations that may induce sleepiness. The score range is 0–24 where a score of more than 10 is regarded as excessive daytime sleepiness .Finally, HAD was used to detect and evaluate symptoms of anxiety (HAD-A) and/or depression (HAD-D). These scales have seven items with a rating from 0 to 3 and a total score of 21 where a score above 8 indicates the presence of anxiety or depression .The skewness ratio  was used to check for normality in the outcome instruments for each treatment group at all three session points. Only ISI and ESS at baseline for the AA group showed a skewness ratio exceeding ±2 indicating that the majority of measures showed satisfying normality. To examine treatment effects over time a series of linear mixed models, using the Restricted Maximum Likelihood method and unstructured variance components, were estimated with the ISI, ESS, DBAS-16, and HAD questionnaires as outcome variables. Treatment group, gender, session point, time on medication, age, and the interaction of treatment group and session point were chosen as fixed factors/covariates. Dropouts were assessed using Pearson Chi-Square test and Mann-Whitney test. A significance level of 5% was used. All analyses were performed with SPSS 21.The groups did not differ significantly from each other on ISI at baseline measurements; nor were there any differences between the participants and the dropouts. In total 59 participants were analysed at baseline, 27 in the AA group and 32 in the CBT-i group.NCT01765959).The project was approved by the Regional Ethical Vetting Board in Uppsala (ref number 2012/353) and was registered in the ClinicalTrials.gov database , and at the 6-month follow-up (p < 0.05). In DBAS-16 there was a significant change in favour of the CBT-i group at the posttreatment measurement point (p < 0.01); however the result was not maintained at the 6-month follow-up. The between-group parameter estimate (PE) after treatment and at the 6-month follow-up was 6.28 (p < 0.001) and 2.95 (p < 0.05) for the ISI and 1.51 (p < 0.01) and 1.29 (p < 0.05), respectively, for DBAS-16. For HAD-D there were no differences between the groups since both groups showed significant improvements. None of the groups showed improvements in HAD-A or ESS. Comparisons between the groups are presented in The AA group was compared to the CBT-i group and the results were analysed to reveal changes over time from baseline to posttreatment measurement point and from baseline to the 6-month follow-up. Significant between-group improvements occurred in favour of CBT-i in the primary outcome measure, ISI, at the posttreatment measurement point  and from baseline to the 6-month follow-up . The PE for ISI were −8.16 (p < 0.001) after treatment and −6.09 (p < 0.001) at the 6-month follow-up. For DBAS-16 the PE were −1.80 (p < 0.001) after treatment and −1.76 (p < 0.001) at the 6-month follow-up.ISI and DBAS-16 scores declined from baseline to posttreatment measurement point (p < 0.05), PE −0.99 (p < 0.05). No improvements were seen in HAD-A or ESS.Clinically significant posttreatment improvements measured by ISI showed that the CBT-i group had three (12%) cases with moderate improvement and thirteen (52%) cases with marked improvement. At the 6-month follow-up there were two (9%) cases with moderate improvement and nine (39%) cases with marked improvement. HAD-D scores also declined significantly from baseline to the 6-month follow-up (p < 0.05), PE −2.07 (p < 0.05), and at the 6-month follow-up (p < 0.001), PE −3.27 (p < 0.001). Clinically significant posttreatment improvements measured by ISI showed zero cases of moderate improvement and two (8%) cases of marked improvement. At the 6-month follow-up there was one (5%) case with moderate improvement and there were three (14%) cases with marked improvement. HAD-D scores declined significantly from baseline to the 6-month follow-up (p < 0.05), PE −0.70 (p < 0.05). No significant changes were seen in DBAS-16, HAD-A, or ESS.ISI scores declined at the posttreatment measurement point  as suggested by Morin et al. . In the Dysfunctional beliefs concerning sleep, sleep medication, and consequences of insomnia are a common feature for persons suffering from insomnia disorder and traditionally the DBAS-16 is an effective measure for CBT-i outcomes. CBT requires questioning one's behaviour in order to achieve behavioural changes. In accordance with previous findings , 38, 39,As mentioned previously, insomnia disorder may give rise to anxiety and/or depression. These disorders may also give rise to insomnia disorder. In this study HAD was not used to diagnose anxiety and/or depression but to detect the occurrence of symptoms thereof. Both groups showed a statistically significant reduction of depressive symptoms measured by HAD-D six months after the intervention, but as a group the participants were not classified as clinically depressed.Small sample sizes are always an issue and are indeed a limitation. This should be kept in mind when interpreting the results. In addition, this study contains a low number of male participants, which is a limitation in the sense that there is no way to detect potential gender differences in the sample. Another limitation may be that no measurements of the treatment preferences and expectations were performed. This would have been of value since there were participants in both groups who were dissatisfied with the group allocation, which may have affected the credibility of the treatment results. One further limitation was that the CBT-i and the AA were administered in different dosages as regards time spent in therapy.In this study auricular acupuncture was compared to CBT-i, which is regarded as the most effective nonpharmacological treatment for insomnia disorder. Compared to CBT-i, auricular acupuncture, as offered in this study, cannot be considered an effective stand-alone treatment for persons with insomnia disorder.